The objective of this project is to characterize the functional role of protein O- GlcNAcylation in crucial areas of stem cell biology. O-GlcNAcylation is a post- translational modification in which a single monosaccharide (O-GlcNAc) is installed on Ser or Thr residues of cytosolic and nuclear proteins. This modification has been found on a wide array of proteins, including transcription factors. Although much is known about the crucial role of O-GlcNAcylation in cellular processes, this modification has yet to be studied in the context of regenerative medicine. I propose herein to characterize the functional role of O- GlcNAcylation in two critical areas of stem cell biology: neuronal differentiation (Aim 1), and the repression of genomic transcripts by polycomb group proteins (Aim 2). In Aim 1, I propose to use Metabolic Oligosaccharide Engineering (MOE) and glycoproteomic analysis to identify the protein substrates of O-GlcNAcylation present during various stages of neuronal differentiation. These studies will aid in the elucidation of the molecular determinants of stem cell fate decisions during neuronal differentiation. In Aim 2 I will study the putative role of O-GlcNAcylation in the maintenance of hESC and adult stem cells by polycomb group proteins (PcGs). Recent work in Drosophila has shown that O-GlcNAcylation governs PcG-mediated gene silencing during development. Herein we will identify both O-GlcNAcylated proteins and the genes that are being modulated by them by performing a chromatin immunoprecipitation (ChIP)-type assay facilitated by MOE. This work may expand the current understanding of the molecular mechanisms underlying self-renewal and pluripotency of hESCs. In summary, the successful completion of the studies proposed herein will constitute the first-ever comprehensive study of protein O-GlcNAcylation in hESCs, and will greatly expand the current understanding of molecular mechanisms governing neuronal differentiation, pluripotency and transcriptional control in hESC biology.